摘要
本研究鉴定了鱼类重要致病菌杀鱼爱德华氏菌(Edwardsiella piscicida)抗强酸系统GadBCD及其功能。通过序列分析和共转录实验表明:GadBCD系统由1个谷氨酸脱羧酶、1个谷氨酰胺酶和3个转运体组成,它们组成1个操纵子。 qRT-PCR发现强酸和高温刺激细菌时gadBCD表达基本不变,但过氧化氢和宿主血清刺激时gadBCD表达显著上调。利用同源重组构建了GadBCD系统缺失突变株ΔgadP,通过比较野生株和ΔgadP的生长曲线以及酸性压力下存活率等实验,发现GadBCD系统不仅是杀鱼爱德华氏菌抗中强酸的重要参与者,也是抗强酸所必需的;通过比较野生株和ΔgadP在生物膜形成、运动性、抗宿主血清杀伤、感染细胞等方面的差异,发现GadBCD参与了细菌的毒力。综上所述, GadBCD系统是杀鱼爱德华氏菌重要的抗强酸系统,并参与了细菌的致病作用。
杀鱼爱德华氏菌(Edwardsiella piscicida)属肠杆菌科(Enterobacteriaceae)爱德华氏菌属(Edwardsiella),曾又称迟缓爱德华氏菌(E. tarda)。 ABAYNEH
病原菌经常不可避免会遇到各种各样的环境压力,如高温、高渗透压、氧化应激、pH波动、饥荒等。在这些环境压力中,低pH往往是病原体最常见的困
谷氨酸依赖型抗酸系统主要是利用谷氨酸脱羧酶催化的脱羧反应消耗
在前期研究中,我们鉴定了杀鱼爱德华氏菌抗弱酸系统CadB
杀鱼爱德华氏菌(E. piscicida TX01)分离自患病的鱼
LB培养基(g/L):胰蛋白胨 10、酵母提取物5、NaCl 10,pH 为9.0、7.0、5.0、4.5、4.2、3.0和2.5等。牙鲆腮细胞系(FG 细胞)和小鼠巨噬细胞(RAW264.7 细胞)由中国科学院海洋研究所馈赠。
酵母浸粉、胰蛋白胨购自Oxoid公司;T4连接酶购自宝生物工程(大连)有限公司;质粒提取试剂盒、胶回收试剂盒、RNA提取试剂盒购自OMEGA公司;琼脂粉、多黏菌素B、氯霉素和氨苄青霉素购自北京索莱宝科技有限公司;逆转录试剂盒购自Invitrogen公司;DMEM细胞培养液购自Corning公司;L-15细胞培养液购自吉诺生物工程有限责任公司;胎牛血清、胰蛋白酶购自Thermo Fisher公司;PBS缓冲液、PCR Master MIX购自生工生物工程(上海)股份有限公司; qPCR Super MIX购自北京全式金生物技术股份有限公司。本研究所用引物见
引物 Primer | 引物序列(5'-3') Sequence (5'-3') | 用途 Function |
---|---|---|
GadPKOF1 | ggatccATGTTGGGCTTATCCACCGA (BamHⅠ) | 基因敲除 |
GadPKOR1 | TAGCGTTACGGCGCATAACGACG | 基因敲除 |
GadPKOF2 | ATGCGCCGTAACGCTAACTAATAGATAAGAA | 基因敲除 |
GadPKOR2 | ggatccCAACATCCTGGCGCTGAA (BamHⅠ) | 基因敲除 |
GadPKOF3 | GCCTCTGAACCAAAGCG | 基因敲除 |
GadPKOR3 | CCGTTCGGGTACGATGT | 基因敲除 |
GadC3-GadC2RTF | CGGGGGACAAAACATCGACA | RT-qPCR |
GadC3-GadC2RTR | CTGGTGATGCTGCTGGTGAT | RT-qPCR |
GadC2-GadDRTF | CAAAGACAAAGGCCAGCCAG | RT-qPCR |
GadC2-GadDRTR | GAAAAATGGTGGCGGACGTC | RT-qPCR |
GadD-GadCRTF | GGCCAGCGTCGCATACTG | RT-qPCR |
GadD-GadCRTR | GGCCCAGGCACAGCAGA | RT-qPCR |
GadC-GadBRTF | CTGACCACCGCGACGATAT | RT-qPCR |
GadC-GadBRTR | CCAGCTGCTGATGGAGGACTT | RT-qPCR |
ETAE-2863RTF | GGTAACTGTAGCACGGCCA | RT-qPCR |
ETAE-2863RTR | GTCACCATAATGATCGGCATC | RT-qPCR |
ETAE-2869RTF | TTTATGATCGATCGCAGCAA | RT-qPCR |
ETAE-2869RTR | GCATCAGAAAGCGAAAGTGTT | RT-qPCR |
在NCBI数据库中获取杀鱼爱德华氏菌GadBCD系统5个蛋白(GadB、GadD、GadC、GadC2和GadC3)的氨基酸序列,用DNAMAN 5.0 软件对该系统5个蛋白的氨基酸序列进行比对分析;用在线软件对5个蛋白质的理化性质、跨膜结构、保守结构域和三维结构等进行在线预测分析。
将杀鱼爱德华氏菌TX01接种于 LB 液体培养基中,28 ℃ 培养至OD600=0.8;离心收集菌体,用细菌基因组DNA提取试剂盒和HP Total RNA kit分别提取基因组DNA(gDNA)和总RNA,RNA 反转录cDNA。分别以cDNA、gDNA和RNA(阴性对照)为模板,用特异性引物进行PCR扩增。PCR反应体系:模板1 μL、引物F/R 0.5 μL、2×PCR Mix 6.25 μL、加超纯水至20 μL。反应条件:94 ℃ 5 min;94 ℃ 30 s,57 ℃ 30 s,72 ℃ 30 s,35个循环;72 ℃ 5 min。实验重复3次。
将培养至对数期的杀鱼爱德华氏菌TX01在 pH 2.5 、42 ℃、血清和过氧化氢的条件下刺激1 h,提取总RNA反转cDNA,进行 RT-qPCR 检测基因的表达。RT-qPCR 反应体系(20 μL):SYB
由于GadBCD系统所属的5个基因阅读框过长,难以进行缺失突变,因而我们考虑缺失GadBCD系统启动子区域209 bp的一段序列。引物的核苷酸序列见
提取野生株TX01和敲除株ΔgadP总RNA,反转录为cDNA,通过RT-qPCR检测gadBCD的表达水平,以确认敲除株ΔgadP中gadBCD不表达或超低水平表达。
将对数期的野生株和敲除株ΔgadP稀释至1
将对数期的野生株和敲除株稀释100倍,在pH=7、pH=2.5和pH=2.5且添加1 mmol/L Glu 3种条件下的PBS缓冲液中孵育2 h,细菌经系列梯度稀释后涂布在含有polyB的LB平板上进行菌落计数。实验重复3次。
细菌生物膜形成能力的检测采用结晶紫染色法,具体方法参考MA
细菌运动性分析参考HUO
细菌对牙鲆FG 细胞的黏附和侵染实验参考JIN
细菌在RAW264.7细胞中的增殖按XIE
细菌在罗非鱼血清的存活率实验按FANG
前期在对杀鱼爱德华氏菌抗酸性能研究中,鉴定了1个抗弱酸系统CadBA。然而杀鱼爱德华氏菌抗强酸系统还完全未知。通过分析杀鱼爱德华氏菌基因组序列,我们发现了1个疑似谷氨酸脱羧系统,命名为GadBCD。GadBCD系统由GadB、GadC、GadD、GadC2和GadC3共5个蛋白组成。GadB(ETAE_2868)开放阅读框(ORF)由1 395 bp组成,编码464个氨基酸,与大肠杆菌的谷氨酸脱羧酶GadB具有79%的一致性,具有脱羧酶的保守结构域,三维结构呈六聚体(
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图1 杀鱼爱德华氏菌GadBCD系统5个组成的结构域分析与三维结构预测
Fig.1 Structural domain analysis and tertiary sgructure prediction of E. piscicida GadBCD system
在许多细菌如大肠杆菌和单增李斯特菌中,gadB 和gadC在1个操纵子下进行共转录,以快速的应对胞内外强酸压力。在E. piscicida中,gadC3和gadC2之间有1个15 bp的基因重叠区,gadC2和gadD之间有1个6 bp的基因间隔区,gadD和gadC之间有1个76 bp的基因间隔区, gadC和gadB之间有1个37 bp的基因间隔区(
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图2 杀鱼爱德华氏菌GadBCD操纵子示意图和共转录验证
Fig.2 Schematic organization and co-transcriptional verifcation of the GadBCD operon in E. piscicida
S1和S2表示预测的2个转录起始位点,F和R为引物。
S1 and S2 denote the predicted transcription start site, F and R are primers.
上述结果表明,杀鱼爱德华氏菌GadBCD系统由5个蛋白组成,且谷氨酸脱羧酶和谷氨酰胺酶连同3个转运蛋白组成同1个操纵子。与其他细菌谷氨酸脱羧系统相比,杀鱼爱德华氏菌GadBCD系统具有组成的独特性和新颖性。
在酸性压力环境下细菌抗酸基因往往诱导表达。为探究GadBCD表达是否受外界环境的影响,将杀鱼爱德华氏菌在酸压力、氧化压力、宿主血清和高温等逆境条件下刺激1 h,RT-qPCR分析刺激前后GadBCD表达水平。由于GadBCD系统内各个基因是共转录关系,选择检测gadB的表达来替代GadBCD的表达水平。结果显示,相比于正常条件下,gadB表达在强酸和高温刺激下基本不变,而在过氧化氢和血清刺激时分别上调了7倍和5倍(
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图3 GadBCD系统表达分析
Fig.3 Expression of GadBCD system
(a)逆境条件下gadBCD的表达分析;虚线代表pH = 7为对照组;(b)ΔgadP中gadBCD、ETAE_2863和ETAE_2869的表达分析;虚线代表野生株的表达为对照组。
(a)The gadBCD expression of TX01 under different adversity. Dashed line represents control(pH = 7);(b) The expression of gadBCD, ETAE_2863 and ETAE_2869 in ΔgadP. Dashed line represents the expression of the wild strain as a control.
框内基因缺失是研究基因功能的首选方法,考虑到GadBCD系统有5个基因组成,且全长有5 962 bp,全部基因的缺失难以获得。为此,我们缺失了GadBCD系统上游启动子209 bp区域,获得的敲除株命名为ΔgadP。为验证敲除后的效果,RT-qPCR比较了野生株和敲除株GadBCD系统表达的差异。结果显示,与野生株相比,敲除株ΔgadP中gadBCD的表达下调约1 200倍,而GadBCD系统邻近的上下游基因ETAE_2863和ETAE_2869的表达不变(
为探明GadBCD系统是否在杀鱼爱德华氏菌抗酸过程中起作用,我们将野生株TX01和敲除株ΔgadP在不同酸性环境下生长(4.2 < pH ≤7.0),并检测细菌的生长状况。结果表明,在中性pH,即pH = 7.0时,野生株TX01和敲除株ΔgadP的生长一致(
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图4 杀鱼爱德华氏菌在不同条件下的生长状态
Fig.4 Growth analysis of E. piscicida in diferent conditions
为明确GadBCD系统是否在杀鱼爱德华氏菌抗强酸过程中起作用,我们将对数期的野生株TX01和敲除株ΔgadP在强酸(pH=2.5)的环境中挑战2 h,然后检测存活的细菌。结果表明,野生株TX01的存活率为8.3%,而敲除株ΔgadP的存活率仅为0.5%,显著低于野生株(
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图5 酸性条件下的存活率
Fig.5 Survival rate under acid condition
在明确GadBCD系统与杀鱼爱德华氏菌耐酸性能的关系后,我们继续探究该系统是否与细菌其他性能如生物膜形成和运动性相关。为此,利用结晶紫染色(CV)法检测了杀鱼爱德华氏菌生物膜的形成。结果显示,敲除株ΔgadP 的生物膜形成能力明显强于野生株TX01(
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图6 GadBCD突变对生物膜形成和运动性的影响
Fig.6 Effects of GadBCD mutations on bioflm formation and mutations on motility
为检测GadBCD系统对运动性的影响,野生株TX01和敲除株ΔgadP穿刺接种到软琼脂运动性平板内,培养18 h 后发现,敲除株ΔgadP 的游动圈直径显著小于野生株TX01(
既然GadBCD系统与细菌的抗酸性、生物膜形成和运动性等性能相关,推测其很可能参与细菌的致病性。为明确GadBCD系统是否在杀鱼爱德华氏菌致病过程中起作用,我们将对数期的野生株TX01和敲除株ΔgadP分别感染 FG 细胞1 h 和2 h,然后检测黏附到细胞表面和侵入到细胞内的细菌。结果表明,在2个检测时间点,均发现敲除株ΔgadP的数量显著低于野生株TX01(
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图7 GadBCD系统对杀鱼爱德华氏菌致病性影响
Fig.7 Effects of GadBCD on pathogenicity of Edwardsiella piscicida
(a)对FG细胞的黏附和侵袭;(b) 巨噬细胞RAW264.7中的存活和复制;(c) 血清压力下的存活率。
(a)The adhesion and invasion to FG cells;(b) Survival and replication in macrophage cell RAW264.7;(c)survival rate under serum pressure.
为检测GadBCD系统对细菌在宿主吞噬细胞内存活的影响,野生株和敲除株感染小鼠巨噬细胞 RAW264.7,杀灭胞外细菌后检测胞内存活的细菌数。结果表明,在4个检测感染时间点,巨噬细胞内存活的ΔgadP数量均明显少于野生株TX01(
杀鱼爱德华氏菌具有强的抗宿主血清杀菌能力,可通过血液引发系统性感染。为探究GadBCD系统与细菌抗宿主血清杀菌能力的关系,野生株和敲除株与罗非鱼血清孵育1 h后检测存活细菌的数量。结果表明,敲除株ΔgadP在宿主血清中的存活率仅为40%,显著低于野生株80%的存活率(
对于许多动物病原菌来说,对中强酸和强酸的耐受能力决定其能否在宿主胃酸和吞噬细胞等酸性环境中存活,因而耐酸性是病原菌致病的一个重要性能。谷氨酸脱羧酶系统往往是众多致病菌最重要的抗强酸系统。大肠杆菌的谷氨酸脱羧酶系统,由谷氨酸脱羧酶GadA和GadB以及转运蛋白GadC组成,且GadB和GadC处在1个操纵子上;研
杀鱼爱德华氏菌是水产重要致病菌,其致病机制的研究近年来取得积极进展,一些重要的毒力因子和新型的毒力因子相继被鉴定和阐
杀鱼爱德华氏菌GadBCD系统的独特性产生了3种效应:(1)具有抗中强酸作用。本研究结果表明,GadBCD系统的敲除致使杀鱼爱德华氏菌在中强酸(pH=4.2)条件下无法正常生长,而野生株可正常生长。(2)具有抗强酸作用。GadBCD系统缺失导致杀鱼爱德华氏菌在强酸刺激2 h后的存活率从8.3%锐降至0.5%。(3)比其他细菌如大肠杆菌的谷氨酸脱羧酶系统具有更高效的抗酸功能。本研究结果表明杀鱼爱德华氏菌在pH = 2.5酸性环境下刺激2 h的存活率(8.3%)大大高于同等条件下大肠杆菌的存活率(0.2%
细菌的耐酸性与生物膜形成密切相关。生物膜是一种嵌入在胞外聚合物的自生成基质中的微生物聚集集体,微生物通过生物膜黏附或附着在材料表
细菌的生物膜形成和运动性等生理性能与细菌的致病性密切相关,且耐酸性也是一些肠道病原菌致病的重要因素,因此我们分析了GadBCD系统与细菌致病性的关系。发现GadBCD系统的缺失减弱了杀鱼爱德华氏菌的对宿主非吞噬细胞的黏附和侵入、减弱了细菌在宿主吞噬细胞的繁殖和存活能力、降低了细菌抵御宿主血清杀伤的能力,这充分说明GadBCD系统与杀鱼爱德华氏菌毒力密切相关。相似地,大肠杆菌谷氨酸脱羧酶系统也有细菌致病性,SHIN
综上所述,本研究首次在杀鱼爱德华氏菌中鉴定了1个新型的谷氨酸脱羧酶系统GadBCD。GadBCD系统包括了谷氨酸脱羧酶、谷氨酰胺酶以及3个转运体,5个蛋白编码基因组成1个操纵子。该系统不仅是杀鱼爱德华氏菌耐中强酸和强酸所必需的,而且还与细菌生物膜形成和运动性相关,同时也是细菌感染宿主细胞的重要参与者,因而是杀鱼爱德华氏菌1个重要的毒力因子。本研究为了解杀鱼爱德华氏菌的耐酸机制与致病机制提供了新的思路。
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